【作者】 白瑞霞；

【导师】 彭建营； 张玉星；

【作者基本信息】 河北农业大学 ， 果树学， 2005， 硕士

【摘要】 AFLP（扩增片段长度多态性）是一种新的DNA分子标记技术,具有高效、稳定、可靠的特点。广泛应用于品种鉴定、遗传多样性分析、遗传连锁图谱构建等方面。枣是起源于我国的重要果树,品种繁多,分布范围广,遗传背景复杂。枣的分类涉及形态学、孢粉学、生物化学、分子生物学等领域,经历了从形态多样性到DNA多样性的发展过程。由于分类标准不统一、分类手段的局限性,导致分类结果不一致。部分品种来源不清,品种亲缘关系不明,同物异名及同名异物现象十分普遍,不利于枣种质资源的研究和品种的选育与推广。本研究应用银染AFLP技术体系构建了82个枣供试材料的DNA指纹图谱,对枣的种质鉴定、品种分类、亲缘关系、遗传多样性进行了分析,主要研究结果如下: 1.确定了适于AFLP分析的枣基因组DNA提取方法为改良CTAB法,其提取液成份为:100 mmol/L Tris-HCl（pH 8.0）,20 mmol/L EDTA,1.4 mol/L NaCl,2% CTAB,2%PVP₄₀,50 mmol/L β-巯基乙醇,粗提后的DNA经RNase除去RNA;苯酚/氯仿抽提一次,氯仿/异戊醇抽提一次,去除蛋白质,得到了基本无降解,蛋白质和RNA去除彻底,纯度高、质量好的枣基因组DNA。 2.建立了适合枣基因组的AFLP银染技术体系:在20μL酶切体系中,包括基因组DNA 450ng,NEB buffer22μL,BSA（10mg/mL）0.2μL,EcoR Ⅰ（大连宝生物公司）3U,Mse Ⅰ（NEB公司）3U,加ddH₂O至20μL;37℃酶切3h;加上接头,37℃连接10h（或过夜）;连接产物稀释10倍进行预扩增;预扩增产物稀释10倍进行选择性扩增;加入10μL Loading Buffer,95℃变性10min,立即冰浴;在6%变性聚丙烯酰胺凝胶电泳上进行电泳分析;银染法检测PCR产物。 3.采用7对引物组合对82个供试材料进行扩增分析,共扩增出467条电泳谱带,平均每对引物产生约67条谱带,其中94条带为所有材料共有,占20.13%,373条为多态性带,多态性检出率为79.87%,从DNA分子水平显示出枣具有丰富的遗传多样性。 4.用7对引物组合构建了82份供试材料的AFLP指纹,采用二歧分类法可以将供试材料一一区分开,并且部分材料拥有自己的特征带,可以作为该材料鉴定的依据。 5.应用离差平方和法对供试材料的AFLP数据结果进行聚类分析,将82个供试材料分为6类,可作为枣分类的依据和基础。 6.建立了82份供试材料的AFLP树状图,结合各样品间的遗传距离（欧式距离）对其遗传多样性进行了分析,揭示了供试材料间遗传背景的相似性及复杂性。 以上研究为枣基因库的建立、品种资源保护利用、优良品种选育、新品种知识产权保护以及亲缘关系的系列研究奠定了基础,同时为枣分子标记辅助育种提供了可能。更多还原

【Abstract】 As a new DNA marker, AFLP （amplified fragment length polymorphism） has been widely used in cultivars identification, genetic diversity, and genetic linkage mapping in fruit trees. As one of the important fruit trees, Ziziphus jujuba originated from China. It has very long culture practice history, a large number of cultivars, and very wide distribution. The classification evolved from morphological diversity to genetic diversity. The studies on Ziziphus jujuba classification involved the fields of morphology, palynology, biochemistry and molecular biochemistry. Because of the different standard adopted and each kind of classification’s limitation, both synonym and homonym are presented very seriously. In addition, the genetic relationships among cultivars were not very clear. AFLP silver-staining technique system suitable for Ziziphus jujuba was established in this study. The fingerprints of 82 materials were constructed, and the genetic diversity was analysed. The aim was to set up a classification system on Ziziphus jujuba and supply theoretical basis for conservation and utilization as well as technological foundation for cultivars registration, identification and protection. The main results were briefly summarized as follows:1. The genomic DNA of Ziziphus jujuba was extracted by a modified CTAB method. The modified extraction buffer contained 100 mmol/L Tris-HCl （pH 8.0） ; 20 mmol/L EDTA, 1.4mol/L NaCl, 2% CTAB, 2% PVP₄0, and 50 mmol/L P -Mercaptoethanal （added before isolation）. RNA was removed from the solution with RNase, and DNA was purified by phenol / chloroform, chloroform / isoamyl alcohol. The purified genomic DNA was suitable for AFLP analysis.2. AFLP silver-staining analysis system suitable for Ziziphus jujuba genomic DNA was established. 450ng DNA was digested with 3 units of EcoR I and 3 units of Mse I enzymes in a reaction volume of 20 U L and incubated at 37°C for 3h.. Double-stranded adaptors were added to the restriction fragments at 37°C for more than 10 h（or overnight）. The linked products was diluted 10 times with TE buffer and used as templates for pre-amplification. The pre-amplification was carried out using an EcoRI and a Msel site primer, both without selective bases. The pre-amplification products were diluted 10 times更多还原